Advances in instrumentation often lead to conceptual breakthroughs. For instance, laser-based optical tweezers led to experiments on the physical forces involved in stretching and binding of biomolecules. The long-term goal of this exploratory program is to provide a new technology for trapping individual biomolecules in aqueous solution, the Anti-Brownian ELectrophoretic trap (ABEL trap). This device will allow trapping of objects 50-100 times smaller than the smallest objects typically trapped with optical tweezers, extending all the way to single proteins and nucleic acids in solution. [unreadable] [unreadable] The first aim will develop and extend the ABEL trapping concept to enable trapping of individual small, dim biomolecules in buffer, such as a single GFP, a single quantum dot biolabel, and a single molecule of the chaperonin GroEL labeled with only one fluorophore. This will involve reduction of latency by using hardware feedback and optimization of the microfluidic design and surface treatment. A second aim will develop analysis procedures that will allow extraction of the mobility and diffusion coefficient of the single trapped biomolecule as a function of time. [unreadable] [unreadable] The ABEL trap will enable extended study of single biomolecules in free solution-a capability which has not previously existed. The ABEL trap directly provides information on the transport properties of the trapped object, such as the occurrence of protein-protein interaction events. In future work, this capability will lead to study of aggregate formation as monomers are added one by one, and eventually the ABEL trap should shed light on the initial events that lead to aggregation and/or misfolding of prions or other proteins in diseases that arise from these abnormalities. [unreadable] [unreadable] This project will also introduce a new concept into single-biomolecule research: that feedback can be used to cause a single molecule (or small collection of molecules) to violate the Second Law, while preserving the Second Law on the macroscale. The Project Description details the physical, chemical, and biophysical experiments that are required to achieve the main aims; but once the instrumentation is availble to the biomedical community, others will think of experiments that cannot be imagined at the present. It is for this reason that this research represents a new and powerful potential advance for biomedical applications. [unreadable] [unreadable] [unreadable]